Anti tbcb

Western blot analysis was performed as previously described 36 (link). The cells were washed with pre-chilled PBS and lysed with 1 × SDS-PAGE sample loading buffer (#E153-01, GenStar, China) at room temperature (RT) for 30 minutes, followed by boiling for 10 minutes at 100°C. The proteins were separated on 12.5% SDS-PAGE gels (PG113, Epizyme Biotech, China) and transferred to 0.2-μm polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Germany). The membranes were blocked with 3% nonfat dry milk at RT for one hour and incubated overnight at 4°C with primary antibodies at the appropriate dilutions. Subsequently, they were incubated with secondary antibodies for one hour at RT and detected by a Bio-Rad ChemiDoc (Bio-Rad, USA). The primary antibodies were used in this study as follow: anti-TBCB (A13248, Abclonal, China) and anti-β-Actin (3700S, Cell signaling technology, USA). All primary antibodies were used at a 1:1000 dilution, and the secondary antibodies (Jackson ImmunoResearch, USA) were used at a 1:10000 dilution.

The astrocytes were fixed in −20° precooled acetone and methanol (1:1) for 5 min and then blocked with 5% bovine serum albumin (BSA) at room temperature for 30 min. The astrocytes were probed with the indicated primary antibodies (anti-TBCB, 1:50, A13248, ABclonal, China; anti-TBCB, 1:250, sc-377139, Santa Cruz, USA; anti-α- tubulin, 1:5000, GTX628802, GeneTex, Irvine, California, USA) properly diluted at 4° overnight. Then, the cells were incubated with secondary antibodies (FITC goat anti-rabbit IgG, 1:200, E031220-01, EARTH, China; Cy3 goat anti-mouse IgG, 1:200, Abbkine, Wuhan, China) and stained with DAPI (C1005, Beyotime, Shanghai, China). Subsequently, the cells were mounted in Fluorescence Mounting Medium (ab104135, Abcam, Cambridge, UK) and sealed with nail polish. Images were obtained by confocal laser scanning microscopy (Leica DMI8, Germany) and the intensity of the fluorescence was analysed by ImageJ (1.53 c) software (n = 6) [53 (link),55 (link)]. The astrocyte processes in the high magnification images were also counted by ImageJ (1.53 c) software (approximately 50 cells were counted in each group). IF was mainly used to observe the changes in cell morphology and protein distribution in this study.